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Gene cloning, expression, and characterization of a novel acetaldehyde dehydrogenase from Issatchenkia terricola strain XJ-2

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Abstract

Acetaldehyde is a known mutagen and carcinogen. Active aldehyde dehydrogenase (ALDH) represents an important mechanism for acetaldehyde detoxification. A yeast strain XJ-2 isolated from grape samples was found to produce acetaldehyde dehydrogenase with a high activity of 2.28 U/mg and identified as Issatchenkia terricola. The enzyme activity was validated by oxidizing acetaldehyde to acetate with NAD+ as coenzyme based on the headspace gas chromatography analysis. A novel acetaldehyde dehydrogenase gene (ist-ALD) was cloned by combining SiteFinding-PCR and self-formed adaptor PCR. The ist-ALD gene comprised an open reading frame of 1,578 bp and encoded a protein of 525 amino acids. The predicted protein of ist-ALD showed the highest identity (73%) to ALDH from Pichia angusta. The ist-ALD gene was expressed in Escherichia coli, and the gene product (ist-ALDH) presented a productivity of 442.3 U/mL cells. The purified ist-ALDH was a homotetramer of 232 kDa consisting of 57 kDa-subunit according to the SDS-PAGE and native PAGE analysis. Ist-ALDH exhibited the optimal activity at pH 9.0 and 40°C, respectively. The activity of ist-ALDH was enhanced by K+, NH4+, dithiothreitol, and 2-mercaptoethanol but strongly inhibited by Ag+, Hg2+, Cu2+, and phenylmethyl sulfonylfluoride. In the presence of NAD+, ist-ALDH could oxidize many aliphatic, aromatic, and heterocyclic aldehydes, preferably acetaldehyde. Kinetic study revealed that ist-ALDH had a k cat value of 27.71/s and a k cat/K m value of 26.80 × 103/(mol s) on acetaldehyde, demonstrating ist-ALDH, a catalytically active enzyme by comparing with other ALDHs. These studies indicated that ist-ALDH was a potential enzymatic product for acetaldehyde detoxification.

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  1. College of Food Science and Technology, Nanjing Agricultural University, Nanjing, 210095, Jiangsu, People’s Republic of China

    Zhengying Yao, Chong Zhang, Fengxia Lu, Xiaomei Bie & Zhaoxin Lu

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  1. Zhengying Yao
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  2. Chong Zhang
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  4. Xiaomei Bie
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Correspondence to Zhaoxin Lu.

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Yao, Z., Zhang, C., Lu, F. et al. Gene cloning, expression, and characterization of a novel acetaldehyde dehydrogenase from Issatchenkia terricola strain XJ-2. Appl Microbiol Biotechnol 93, 1999–2009 (2012). https://doi.org/10.1007/s00253-011-3541-7

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